MAML1: a coregulator that alters endometrial epithelial cell adhesive capacity

Abstract

Background Abnormalities in endometrial receptivity has been identified as a major barrier to successful embryo implantation. Endometrial receptivity refers to the conformational and biochemical changes occurring in the endometrial epithelial layer which make it adhesive and receptive to blastocyst attachment. This takes place during the mid-secretory phase of woman’s menstrual cycle and is a result of a delicate interplay between numerous hormones, cytokines and other factors. Outside of this window, the endometrium is refractory to an implanting blastocyst. It has been shown that Notch ligands and receptors are dysregulated in the endometrium of infertile women. Mastermind Like Transcriptional Coactivator 1 (MAML1) is a known coactivator of the Notch signaling pathway. This study aimed to determine the role of MAML1 in regulating endometrial receptivity. Methods The expression and localization of MAML1 in the fertile human endometrium (non-receptive proliferative phase versus receptive mid-secretory phase) were determined by immunohistochemistry. Ishikawa cells were used as an endometrial epithelial model to investigate the functional consequences of MAML1 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids. After MAML1 knockdown in Ishikawa cells, the expression of endometrial receptivity markers and Notch dependent and independent pathway members were assessed by qPCR. Two-tailed unpaired or paired student’s t-test were used for statistical analysis with a significance threshold of P < 0.05. Results MAML1 was localized in the luminal epithelium, glandular epithelium and stroma of human endometrium and the increased expression identified in the mid-secretory phase was restricted only to the luminal epithelium (P < 0.05). Functional analysis using Ishikawa cells demonstrated that knockdown of MAML1 significantly reduced epithelial adhesive capacity (P < 0.01) to HTR8/SVneo (trophoblast cell line) spheroids compared to control. MAML1 knockdown significantly affected the expression of classical receptivity markers (SPP1, DPP4) and this response was not directly via hormone receptors. The expression level of Hippo pathway target Ankyrin repeat domain-containing protein 1 (ANKRD1) was also affected after MAML1 knockdown in Ishikawa cells. Conclusion Our data strongly suggest that MAML1 is involved in regulating the endometrial adhesive capacity and may facilitate embryo attachment, either directly or indirectly through the Notch signaling pathway.

Jagged1 regulates endometrial receptivity in both humans and mice

“…51 Our recent study has revealed that knockdown of MAML1 in Ishikawa cells impairs their adhesive capacity. 28 This alteration in adhesion likely does not occur via alterations in the levels of the steroid hormone receptors PGR and ESR1 which is consistent with our observations in JAG1 siRNAtreated Ishikawa cells. Both JAG1 and MAML1 siRNA treatments only reduced the expression of HEY1, but not HES1, 28 indicating similar downstream effects of the Notchdependent pathway.…”

“…28 This alteration in adhesion likely does not occur via alterations in the levels of the steroid hormone receptors PGR and ESR1 which is consistent with our observations in JAG1 siRNAtreated Ishikawa cells. Both JAG1 and MAML1 siRNA treatments only reduced the expression of HEY1, but not HES1, 28 indicating similar downstream effects of the Notchdependent pathway. To investigate whether the regulation of receptivity markers by JAG1 was via the Notch-dependent or -independent pathway, MAML1 siRNA was transfected into Ishikawa cells to determine the effects on the expression of genes associated with receptivity.…”

“…Among the five receptivity markers that were regulated by JAG1, only SPP1 mRNA expression levels were significantly reduced by MAML1 knockdown compared to control ( P < .05; Figure 2F). 28 …”

“…Spheroids were generated using HTR8/SVneo trophoblast cells to mimic blastocyst attachment, as we previously published 26‐28 . Briefly, 2000 HTR8/SVneo cells/well were plated into wells of a U‐shaped, ultra‐low attachment 96‐well plate.…”

“…Spheroids were generated using HTR8/SVneo trophoblast cells to mimic blastocyst attachment, as we previously published. [26][27][28] Briefly, 2000 HTR8/SVneo cells/well were plated into wells of a U-shaped, ultra-low attachment 96-well plate. Cells were then cultured for 48 hours to form spheroids before spheroids were collected and transferred to transfected HEEC or Ishikawa cell monolayer (20 spheroids/well of 96-well plate) to initiate spheroid adhesion assay.…”